The human gut microbiota is a metabolic organ whose cellular composition is determined by a dynamic process of selection and competition. Variation in microbial community composition influences our capacity for energy harvest, sensitivity to infection, processing of xenobiotics, and is implicated in Crohn's disease and other metabolic disorders. This project explores an unexpected finding: a family of small molecules related to vitamin B12 (collectively termed corrinoids) plays a key role in mediating competition and selection between human gut microbes in vivo. My proposed experiments integrate gnotobiotic mouse models, defined consortia of sequenced human gut-derived bacterial symbionts, and a new technique for identifying microbial genes required for gut colonization to address an important prediction: if the production, modification, and consumption of this diverse menu of small molecules influences the composition and functional properties of the human gut microbiota, it may be possible to diagnose and ameliorate pathophysiologic states involving the microbiota by directed manipulation of corrinoid metabolism and/or their molecular targets. Although studied for decades as a critical cofactor for human enzymes, B12 is the only vitamin that is synthesized exclusively by microbes: the role of the diverse corrinoids produced by the human gut microbiota is unexplored. This proposal presents a plan to dissect the role of corrinoid exchange in human gut-associated bacterial symbionts through two Specific Aims. Aim 1 will determine the response of a prominent human gut symbiont (Bacteroides thetaiotaomicron) to exogenous corrinoids. A significant portion of the human gut microbiota lacks the ability to synthesize these small molecules;B. thetaiotaomicron provides a model for determining how such symbionts sense and respond to corrinoids in vivo. My preliminary studies show that B. thetaiotaomicron fitness in the distal gut hinges upon one of the three corrinoid-responsive loci encoded in its genome, and that this requirement is modulated by community context. These studies integrate genetic and biochemical approaches and will establish whether a human gut symbiont can distinguish between different corrinoids, will produce an experimental foundation for identifying and interpreting corrinoid-responsive loci in other genomes, and will provide microbial biomarkers of the corrinoid landscape in vivo. In the distal gut, corrinoids can come from two sources: local biosynthesis by gut microbes and dietary consumption. Aim 2 will define the role of dietary corrinoid consumption on microbiota structure and function in vivo. Gnotobiotic mice, colonized with defined microbial consortia whose complete genome sequences are known, provide a unique opportunity to determine the influence of diet on corrinoid exchange. This influence will be measured through analysis of the effect of dietary B12 consumption on microbial community structure, gene expression, fitness determinants, and corrinoid production. Results from defined microbial communities will be compared with culture-independent studies of a transplanted unfractionated human gut microbiota in gnotobiotic mice that vary in their dietary B12 consumption. Together, these experiments will produce a mechanistic understanding of how the exchange of small molecules is influenced by community composition and host diet to shape the structure and function of the human gut microbiota. My training is in ecology and evolutionary biology (B.A., Princeton University) and microbiology and molecular genetics (Ph.D., Harvard University). My preliminary studies, recently published in Cell Host and Microbe, describe a quantitative massively parallel sequencing technique that allowed the first genetic screen to dissect the establishment of a human symbiont in a mammalian host. The experiments described in this proposal build on some unexpected results from this work, and integrate two key training areas (culture-independent microbial ecology and gnotobiotic animal husbandry) that will expand the range of questions I can pursue in my career goal as an independent investigator. The Gordon lab is an ideal training environment to gain this expertise: the lab is housed in an interdisciplinary Center for Genome Sciences at Washington University and connected with one of the largest university-associated gnotobiotic animal facilities in the country. This proposal also includes a detailed career development plan and timeline that centers on a mentoring committee of two senior and two younger faculty members that I meet with on a regular basis for discussion and advice, three scientific consultants/collaborators who are internationally known experts in these research areas, and the excellent support and mentoring I've received from Jeffrey Gordon. Letters of support from each of these individuals are included in this application. The human gut microbiota contributes an enormous genetic repository distinct from our own in terms of its origin, its composition, and its plasticity: the contribution of our gut microbes to health and disease has received increased attention, but the factors that shape this microbial organ are poorly understood. The proposed experiments will dissect the role of a critical but largely unexplored class of small molecules in this process, and will provide the foundation for tailoring our corrinoid consumption to maximize the contribution of the gut microbiota to human health. PUBLIC HEALTH RELEVANCE: In each of us, microbial cells outnumber our own cells by at least tenfold: our gut microbes encode a dynamic and potentially modifiable gene pool that provides enzymatic functions not encoded in our own genetic material, and much of what we consume, both beneficial and noxious, is encountered and processed by this metabolic organ. Increasing evidence demonstrates that variation in gut microbial community composition has far-reaching consequences for human health, but the factors that shape these communities are poorly understood. This proposal describes experiments to characterize a family of these factors: a group of largely uncharacterized compounds related to vitamin B12 that are exchanged between human gut microbes, and that represent a component of our diet that could be tailored to maximize the contribution of gut microbial communities to human health.